Immunoregulatory compounds and derivatives and methods of treating diseases therewith

ABSTRACT

Compounds are disclosed having the structure of Formula I: 
                         
where R 1 , R 3 , and R 4  are independently hydrogen or C 1  to C 4  alkyl, and R 2  is:
 
                         
where R 5  is selected from the group consisting of hydrogen and C 1  to C 4  alkyl, or
 
                         
where R 6 , R 7  and R 8  are independently hydrogen or C 1  to C 4  alkyl; or the esters or pharmacologically acceptable salts thereof. Such compounds may be utilized for the prophylaxis or treatment of various diseases, particularly inflammatory conditions of the GI tract.
 
     Methods of treating inflammatory conditions of the GI tract such as inflammatory bowel disease using compounds having the following formula are also disclosed: 
                         
where R 9 , R 10  and R 11  are independently selected from the group consisting of hydrogen and C 1  to C 4  alkyl, and R 12  is selected from the group consisting of hydrogen and —C(O)R 13 , where R 13  is a C 1  to C 6  alkyl or an aryl group.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/967,736, filed on Oct. 18, 2004 now U.S. Pat. No. 7,151,095, entitled“IMMUNOREGULATORY COMPOUNDS AND DERIVATIVES AND METHODS OF TREATINGDISEASES THEREWITH”, naming Nnochiri Nkem Ekwuribe and Jennifer A.Riggs-Sauthier as inventors, now allowed, which in turn is acontinuation and claims priority to U.S. patent application Ser. No.10/444,668, filed May 23, 2003, now U.S. Pat. No. 6,903,082, which is adivisional of and claims priority to U.S. patent application Ser. No.09/942,464, filed Aug. 29, 2001, now U.S. Pat. No. 6,583,128, whichclaims priority to U.S. Provisional Application Ser. No. 60/228,683,filed Aug. 29, 2000, the disclosures of each of which are incorporatedherein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to immunoregulatory compounds and methodsof treating diseases therewith.

BACKGROUND OF THE INVENTION

Many people suffer from inflammatory bowel disease (IBD). IBD is ageneric term used to refer to two inflammatory diseases, ulcerativecolitis and Crohn's disease. Ulcerative colitis is a chronicinflammatory disease of unknown etiology that affects various portionsof the gastrointestinal (GI) tract, particularly the lower GI tract, andmore particularly the colon and/or rectum. Crohn's disease is a seriousinflammatory disease of the GI tract. It predominates in the smallintestine (ileum) and the large intestine (colon). Various medicationsare being used to treat inflammatory bowel disease.

It is known to use mesalamine, 5-aminosalicylic acid (5-ASA) to treatulcerative colitis. While mesalamine may be active in treatingulcerative colitis, it may be absorbed as it passes through the GItract. This absorption may adversely affect the amount of mesalaminethat reaches the lower GI tract, particularly the colon and rectum.

Various mesalamine formulations have been introduced in an attempt toprotect mesalamine as it passes through the gut and the upper GI tractOne such formulation is a delayed-release formulation that relies on apH-sensitive coating surrounding the mesalamine. The coating allows themesalamine to pass through the gut and upper GI tract without beingabsorbed so that the mesalamine reaches the target (i.e. the lower GItract, particularly the colon and/or rectum) intact. In anotherformulation, mesalamine microspheres surround a mesalamine core. Thisformulation releases mesalamine throughout the GI tract, rather thantargeting the colon specifically. It may be difficult to predict thebioavailability of the various mesalamine formulations when administeredto a wide variety of individuals. As a result, it may be difficult todetermine the proper dosage for a given individual.

It is also known to use sulfasalazine having the following formula totreat ulcerative colitis.

However, sulfasalazine is metabolized in the body to form mesalamine(5-aminosalicylic acid (5-ASA)) and sulfapyridine. Several adverse sideaffects have been noted from the use of sulfasalazine including nausea,vomiting, abdominal discomfort, and headache to name just a few. Theseadverse side effects are usually attributed to the activity ofsulfapyridine in the GI tract, as well as that absorbed into the system.

U.S. Pat. No. 4,412,992 to Chan proposes mesalamine derivatives. Unlikesulfalazine, the breakdown of these compounds in the intestinal tractmay not give rise to undesirable metabolic products. In fact, thenon-mesalamine metabolic products may be innocuous.

Olsalazine having the following formula has been used to treatulcerative colitis.

In addition to being relatively expensive to make, olsalazine may haveadverse side effects including diarrhea.

It is known to use azathioprine(6-(1-methyl4-nitoimidazol-5-ylthio)purine) in the treatment ofinflammatory bowel disease. Azathioprine has the following chemicalstructure:

It is also known to use 6-mercaptopurine, a metabolite of azathioprine,to treat inflammatory bowel disease. 6-mercaptopurine has the followingchemical structure:

Methotrexate (L4-amino-N¹⁰-methylpteroyl-glutamic acid) has also beenused to treat inflammatory bowel disease. Methotrexate has the followingchemical structure:

The polypeptide cyclosporine, which has traditionally been given totransplant patients to prevent organ rejection, has also been used totreat inflammatory bowel disease. The use of cyclosporine to treat IBDmay be limited, however, by the various side effects associated withthis medication. These side effects include high blood pressure, kidneydamage, tremors, headaches, seizures, excessive hair growth, excessivegum growth confusion, coma, and gout.

SUMMARY OF THE INVENTION

According to embodiments of the present invention, compounds areprovided having the following formula:

where R¹, R³, and R⁴ are independently hydrogen or C₁ to C₄ alkyl, andR² is:

where R⁵ is selected from the group consisting of hydrogen and C₁ to C₄alkyl, or

where R⁶, R⁷ and R⁸ are independently hydrogen or C₁ to C₄ alkyl, aswell as the esters or pharmaceutically acceptable salts of suchcompounds. Pharmaceutical compositions including compounds according toembodiments of the present invention are also provided, as are methodsof treating inflammatory conditions with such compounds.

According to other embodiments of the present invention, methods oftreating an inflammatory condition of the GI tract in a subject in needof such treatment include administering to the subject an effectiveamount of an active pharmaceutical ingredient that includes a compoundof Formula II:

where R⁹, R¹⁰ and R¹¹ are independently selected from the groupconsisting of hydrogen and C₁ to C₄ alkyl; and R¹² is selected from thegroup consisting of hydrogen and —C(O)R where R¹³ is a C₁ to C₆ alkyl oran aryl group, or an ester or a pharmaceutically acceptable salt of suchcompound, in admixture with a solid or liquid pharmaceutical diluent orcarrier. The active pharmaceutical ingredient may further comprise acompound of Formula III:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates embodiments of synthesis routes for compounds of thepresent invention.

FIG. 2 illustrates embodiments of synthesis routes for compounds of thepresent invention.

FIG. 3 illustrates the average reduction in colon:body weight [% BW]utilizing embodiments of the present invention (4-APAA/DNBS andMixture/DNBS) in comparison with results achieved by the prior art(5-ASA/DNBS) and control (vehicle/DNBS).

FIG. 4 illustrates DNBS colitis adhesion scores achieved utilizingembodiments of the present invention (4-APAA/DNBS and Mixture/DNBS) incomparison with results achieved by the prior art (5-ASA/DNBS) andcontrol (Vehicle/DNBS and Vehicle/Sham).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention will now be described with respect to preferredembodiments described herein It should be appreciated however that theseembodiments are for the purpose of illustrating the invention, and arenot to be construed as limiting the scope of the invention as defined bythe claims.

As used herein, the term “inflammatory bowel disease” includesulcerative colitis and Crohn's disease.

According to embodiments of the present invention, compounds areprovided having the following formula:

R¹, R³, and R⁴ are independently hydrogen or C₁ to C₄ alkyl. Preferably,R¹, R³, and R⁴ are independently selected from the group consisting ofH, CH₃, CH₂CH₃, and CH(CH₃)₂. More preferably, R¹, R³, and R⁴ are H orCH₃.

R⁵ is selected from the group consisting of hydrogen and C₁ to C₄ alkyd.Preferably, R⁵ is selected from the group consisting of H, CH₃, CH₂CH₃,and CH(CH₃)₂. More preferably, R⁵ is H or CH₃ and, most preferably, R⁵is H.

R⁶, R⁷ and R⁸ are independently hydrogen or C₁ to C₄ alkyl. Preferably,R⁶, R⁷ and R⁸ are independently selected from the group consisting of H,CH₃, CH₂CH₃, and CH(CH₃)₂. More preferably, R⁶, R⁷ and R⁸ areindependently H or CH₃.

The compounds of the present invention may be made using known startingmaterials and reagents. For example, embodiments of synthesis paths maybe illustrated as shown in FIGS. 1 and 2.

Compounds of the present invention may be utilized for the prophylaxisor treatment of various diseases, particularly inflammatory conditionsof the GI tract including, but not limited to, inflammatory conditionsof the mouth such as mucositis, infectious diseases (e.g., viral,bacterial, and fungal diseases), and Crohn's disease; inflammatoryconditions of the esophogas such as esophagitis, conditions resultingfrom chemical injury (e.g., lye ingestion), gastroesophageal refluxdisease, bile acid reflux, Barrett's esophogas, Crohn's disease, andesophageal stricture; inflammatory conditions of the stomach such asgastritis (e.g., Helicobacter pylori, acid-peptic disease and atrophicgastritis), peptic ulcer disease, pre-cancerous lesions of the stomach,non-ulcer dyspepsia, and Crohn's disease; inflammatory conditions of theintestine such as celiac disease, Crohn's disease, bacterial overgrowth,peptic ulcer disease, and fissures of the intestine; inflammatoryconditions of the colon such as Crohn's disease, ulcerative colitis,infectious colitis (e.g., pseudomembranous colitis such as clostridiumdifficile colitis, salmonella enteritis, shigella infections,yersiniosis, cryptosporidiosis, microsporidial infections, and viralinfections), radiation-induced colitis, colitis in the immunocompromisedhost (e.g., typhlitis), precancerous conditions of the colon (e.g.,dysplasia, inflammatory conditions of the bowel, and colonic polyps),proctitis, inflammation associated with hemorrhoids, proctalgia fugax,and rectal fissures; liver gallbladder and/or bilary tract conditionssuch as cholangitis, sclerosing cholangitis, primary bilary cirrhosis,and cholecystitis; and intestinal abscess. The compounds of the presentinvention may also be utilized in diagnosis of constituents, conditions,or disease states in biological systems or specimens, as well as fordiagnostic purposes in non-physiological systems. Furthermore, thecompounds of the present invention may have application in prophylaxisor treatment of condition(s) or disease state(s) in plant systems. Byway of example, the active component of the conjugate may haveinsecticidal, herbicidal, fungicidal, and/or pesticidal efficacyamenable to usage in various plant systems.

In some embodiments, compounds of the present invention may breakdown inthe intestinal tract to form the metabolic product of Formula IV:

where R¹, R³ and R⁴ are as described above with reference to Formula I,and the metabolic product of Formula V:

The metabolic product of Formula IV may possess anti-inflammatoryactivity and/or immunoregulatory activity. The metabolic product ofFormula V may possess anti-inflammatory activity, and more particularlymay provide inhibition of prostaglandin synthetase I & II.

In other embodiments, compounds of the present invention may breakdownin the intestinal tract to form the metabolic product of Formula IV andthe metabolic product of Formula VI:

where R⁶, R⁷ and R⁸ are as described above with reference to Formula I.The metabolic product of Formula VI may possess anti-inflammatoryactivity and/or immunoregulatory activity. Accordingly, compounds of thepresent invention may provide immunoregulatory activity. Compounds ofthe present invention may also provide inhibition of prostaglandinsynthetase I and II. Compounds of the present invention may be useful intreating various diseases, particularly ulcerative colitis, Crohn'sdisease and the like.

In therapeutic usage, the present invention contemplates a method oftreating an animal subject having or latently susceptible to anintestinal condition(s) or disease state(s) and in need of treatmenttherefor, comprising administering to such animal an effective amount ofa compound of the present invention that is therapeutically effectivefor said condition or disease state. Subjects to be treated by thecompounds of the present invention include both human and non-humananimal (e.g., bird, dog, cat cow, horse) subjects, and are preferablymammalian subjects, and most preferably human subjects.

Depending on the specific condition or disease state to be combatted,animal subjects may be administered compounds of the present inventionat any suitable therapeutically effective and safe dosage, as mayreadily be determined within the skill of the art and without undueexperimentation. For example, compounds of the present invention may beadministered at a dosage between about 0.1 and 100 mg/kg, preferablybetween about 5 and 90 mg/kg, and more preferably between about 10 and80 mg/kg.

The compounds of the present invention may be administered per se aswell as in the form of pharmaceutically acceptable esters, salts, andother physiologically functional derivatives thereof.

The present invention also contemplates pharmaceutical formulations,both for veterinary and for human medical use, which comprise as theactive pharmaceutical ingredient one or more compound(s) of the presentinvention. In such pharmaceutical and medicament formulations, theactive pharmaceutical ingredient preferably is utilized together withone or more pharmaceutically acceptable carrier(s) therefor andoptionally any other therapeutic ingredients. The carrier(s) must bepharmaceutically acceptable in the sense of being compatible with theother ingredients of the formulation and are preferably not undulydeleterious to the recipient thereof. The active pharmaceuticalingredient is provided in an amount effective to achieve the desiredpharmacological effect, as described above, and in a quantityappropriate to achieve the desired daily dose.

The formulations include those suitable for parenteral as well asnon-parenteral administration, and specific administration modalitiesinclude, but are not limited to, oral, rectal, buccal, topical, nasal,ophthalmic, subcutaneous, intramuscular, intravenous, transdermal,intrathecal, intra-articular, intra-arterial, sub-arachnoid, bronchial,lymphatic, vaginal, and intrauterine administration. Formulationssuitable for oral and parenteral administration are preferred, withformulations suitable for oral administration most preferred.

When a compound of the present invention is utilized in a formulationcomprising a liquid solution, the formulation advantageously may beadministered orally or parenterally. When a compound of the presentinvention is employed in a liquid suspension formulation or as a powderin a biocompatible carrier formulation, the formulation may beadvantageously administered orally, rectally, or bronchially.

When a compound of the present invention is utilized directly in theform of a powdered solid, the compound may advantageously beadministered orally. Alternatively, it may be administered bronchially,via nebulization of the powder in a carrier gas, to form a gaseousdispersion of the powder that is inspired by the patient from abreathing circuit comprising a suitable nebulizer device.

The formulations comprising a compound of the present invention mayconveniently be presented in unit dosage forms and may be prepared byany of the methods well known in the art of pharmacy. Such methodsgenerally include the step of bringing a compound of the presentinvention into association with a carrier that constitutes one or moreaccessory ingredients. Typically, the formulations are prepared byuniformly and intimately bringing a compound of the present inventioninto association with a liquid carrier, a finely divided solid carrier,or both, and then, if necessary, shaping the product into dosage formsof the desired formulation.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets,or lozenges, each containing a predetermined amount of a compound of thepresent invention as a powder or granules; or a suspension in an aqueousliquor or a non-aqueous liquid, such as a syrup, an elixir, an emulsion,or a draught.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine, with the active compound being in afree-flowing form such as a powder or granules which optionally is mixedwith a binder, disintegrant, lubricant, inert diluent, surface activeagent, or discharging agent. Molded tablets comprised of a mixture ofthe powdered active compound with a suitable carrier may be made bymolding in a suitable machine.

A syrup may be made by adding a compound of the present invention to aconcentrated aqueous solution of a sugar, for example sucrose, to whichmay also be added any accessory ingredient(s). Such accessoryingredient(s) may include, for example, flavorings, suitablepreservatives, agents to retard crystallization of the sugar, and agentsto increase the solubility of any other ingredient, such as apolyhydroxy alcohol, for example glycerol or sorbitol.

Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of a compound of the presentinvention, which preferably is isotonic with the blood of the recipient(e.g., physiological saline solution). Such formulations may includesuspending agents and thickening agents or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. The formulations may be presented in unit-dose ormulti-dose form.

Nasal spray formulations comprise purified aqueous solutions of acompound of the present invention with preservative agents and isotonicagents. Such formulations are preferably adjusted to a pH and isotonicstate compatible with the nasal mucus membranes.

Formulations for rectal administration may be presented as a suppositorywith a suitable carrier such as cocoa butter, hydrogenated fats, orhydrogenated fatty carboxylic acid.

Ophthalmic formulations are prepared by a similar method to the nasalspray, except that the pH and isotonic factors are preferably adjustedto match that of the eye.

Topical formulations comprise a compound of the present inventiondissolved or suspended in one or more media, such as mineral oil,petroleum, polyhydroxy alcohols, or other bases used for topicalpharmaceutical formulations.

In addition to the aforementioned ingredients, the formulations of thisinvention may further include one or more accessory ingredient(s)selected from diluents, buffers, flavoring agents, disintegrants,surface active agents, thickeners, lubricants, preservatives (includingantioxidants), and the like.

Accordingly, compounds according to the present invention may beutilized for the prophylaxis or treatment of various diseases,particularly diseases of the GI tract including, but not limited to,inflammatory bowel disease.

In still other embodiments of the present invention; methods of treatingor preventing inflammatory bowel disease in a subject in need of suchtreatment or prevention include administering to the subject aneffective amount of an active pharmaceutical ingredient that includes acompound of Formula II:

where R⁹, R¹⁰ and R¹¹ are independently selected from the groupconsisting of hydrogen and C₁ to C₄ alkyl; and R¹² is selected from thegroup consisting of hydrogen and —C(O)R¹³, where R¹³ is a C₁, to C₆alkyl or an aryl group, or an ester or a pharmaceutically acceptablesalt of such compound, in admixture with a pharmaceutical diluent orcarrier.

The active pharmaceutical ingredient may further comprise one or moreother medicaments including, but not limited to, anti-inflammatoryagents such as mesalamine, sulfasalazine, balsalazide, and olsalazine;immunomodulators such as azathioprine, 6-mercaptorpurine, cyclosporineand methotrexate; steroidal compounds such as corticosteroids; andantibiotics such as metronidazole and cirpofloxacin. The activepharmaceutical ingredient preferably further comprises mesalamine, thecompound of Formula III:

When the active pharmaceutical ingredient comprises compounds of FormulaII and Formula III, the compound of Formula II is preferably from about10 to 90 weight percent of the active pharmaceutical ingredient and ismore preferably from about 40 to 60 weight percent of the activepharmaceutical ingredient. When the active pharmaceutical ingredientcomprises compounds of Formula II and Formula III, the molar ratio ofthe compound of Formula I to the compound of Formula II is preferablybetween 1:10 and 10:1, and is more preferably between 1:2 and 2:1.

Subjects to be treated by methods according to these embodiments of thepresent invention include both human and non-human animal (e.g., bird,dog, cat, cow, horse) subjects, and are preferably mammalian subjects,and most preferably human subjects.

Depending on the specific condition or disease state to be combated,animal subjects may be administered the active pharmaceutical ingredientof the present invention at any suitable therapeutically effective andsafe dosage, as may readily be determined within the skill of the artand without undue experimentation. For example, the activepharmaceutical ingredient of the present invention may be administeredat a dosage between about 0.1 and 200 mg/kg, preferably between about 1and 90 mg/kg, and more preferably between about 10 and 80 mg/kg.

The present invention will now be described with reference to thefollowing examples. It should be appreciated that these examples are forthe purposes of illustrating aspects of the present invention, and donot limit the scope of the invention as defined by the claims.

EXAMPLES Examples 1 Through 4 Synthesis of Compounds of the PresentInvention

Melting points were taken on a Laboratory Devices Mel-Temp It capillarymelting point apparatus and are uncorrected. ¹HNMR spectra were obtainedon a Varian Unity 600 MHz spectrometer. Chemical shifts (δ) are reportedas parts per million (ppm) relative to the internal standardtetramethylsilane. Ultraviolet and visible spectra were obtained with aBeckman DU 640i spectrophotometer. Infrared spectroscopy was obtained ona Nicolet Impact 410 and fast atom bombardment (FAB) mass spectroscopydata was obtained by M-Scan Inc. All reagents were used as received fromAldrich Chemical Co.

Examples 1 and 2 Synthesis of5-[4-(1-Carboxy-Ethyl)-Phenylazo]-2-Hydroxy-Benzoic Acid Example 12-(4-Amino-phenyl)-propionic acid

A 500-mL, oven dried, three-neck flask equipped with a stir bar, wascharged with (R,S) 2-(4-nitrophenyl)propionic acid (5.00 g, 25.6 mmol),absolute ethyl alcohol (200 mL), and palladium (10 wt. % on activatedcarbon, 0.27 g, 2.56 mmol). A hydrogen environment was introduced intothe flask and the mixture was then stirred at ambient temperature for 6hours. The crude reaction mixture was filtered through Celite and theethyl alcohol was removed under reduced pressure. The crude product wasdried under vacuum overnight resulting in a light yellow solid(70%-yield, 2.98 g): mp 125-129° C., ¹H NMR (DMSO-d₆): δ 1.24 (3H, s),1.26 (3H, s), 3.41 (1H, s), 3.43 (2H, s), 6.46 (2H, d, J=7.6 Hz), 6.91(2H, d, J=7.6 Hz); IR (KBr), 2596, 2189, 1630, 1581, 1441, 1375, 1277,1192, 1052, 876 cm⁻¹;FAB-MS (NBA), m/z 165 (M+H)⁺.

Example 2 5-[4 (1-Carboxy-Ethyl)-Phenylazo]-2-Hydroxy-Benzoic Acid

As prepared in the above procedure, 2-(4-amino-phenyl)-propionic acid(3.90 g. 23.6 mmol) dissolved in an aqueous HCl solution (75 mL,36.5-38.0% HCl in 8 mL H₂O) was placed in a 200-mL beaker and cooled to0° C. in an ice bath. When the solution was stabilized at 0° C., sodiumnitrite (1.79 g, 26.0 mmol) in water (2 mL) was added dropwise. Thetemperature was maintained at 0-5° C. and the resulting diazonium saltsolution stirred for 15 min.

While the diazonium salt solution stirred, an 800-mL beaker fitted witha stir bar, thermometer, and pH probe (Orion model 420A with Orionsemimicro pH probe) was charged with salicylic acid, sodium salt (11.3g, 20.8 mmol) dissolved in sodium hydroxide (4.25 g, 106 mmol) and H₂O(100 mL). Using an ice bath, the salicylic acid solution was cooled to17° C. and the diazonium salt solution was slowly added in 10 mLportions. Throughout the addition, the pH was maintained at 13.27-13.3with the addition of aqueous sodium hydroxide, and the temperature waskept between 17-18° C. with the addition of ice. After the addition wascomplete, the resulting dark red solution was allowed to warm to ambienttemperature and stirring was continued for 90 min. Upon acidification topH 3.5 with concentrated HCl (˜20 mL, 36.5-38%), a dark red solidprecipitated and was collected by vacuum filtration.

The crude product (8.49 g, 27.0 mmol) was suspended in H₂O (300 mL) andheated at 70° C. for 30 m to remove excess salicylic acid. Thesuspension was cooled to 50° C. and a solid was collected by suctionfiltration. The collected solid was then purified by flashchromatography (SiO₂:ethyl acetate/hexanes, 1:1). The crude product(2.50 g. 7.95 mmol) in DMF (˜4.5 mL) was loaded and yellow coloredfractions were collected, combined, and concentrated under reducedpressure. After drying under vacuum, the purified product was obtainedas an orange solid in 55% yield (1.38 g): mp 147° C., ¹H NMR (DMSO-d₆):δ 1.38 (3H, s), 1.39 (3H, s), 3.76 (1H, s), 3.78 (1H, s), 7.11 (1H, d,J=8.4 Hz), 7.46 (2H, d, J=7.8 Hz), 7.80 (2H, d, J=8.4 Hz), 8.03 (1H, d,J=9.0 Hz), 8.30 (1H, s); IR (KBr), 2973, 1921, 1708, 1652, 1577, 1477,1339, 1289, 1226, 1164, 1101, 1013, 857, 663 cm⁻¹; UV-Vis (MeOH),λ_(max)=355 nm, ε=23,700 mol⁻¹ cm⁻¹ L; FAB-MS (NBA), m/z 313 (M)⁻.

Example 3 Synthesis of 5-(4-Carboxymethyl-Phenylazo)-2-Hydroxy-BenzoicAcid [APAZA]

4-Aminophenylacetic acid (10.0 g, 66.2 mmol) dissolved in an aqueous HClsolution (20 mL, 36.5-38.0%, HCl in 200 mL H₂O) was placed in a 500-mLbeaker and cooled to 0° C. in an ice bath. When the solution wasstabilized at 0° C., sodium nitrite (5.02 g, 72.8 mmol) in water (50 mL)was added slowly in 5 mL portions. The temperature was maintained at0-5° C. and the resulting diazonium salt solution stirred for 15 min.

While the diazonium salt solution stirred, a 2 L beaker fitted with astir bar, thermometer, and pH probe (Orion model 420A with Orionsemimicro pH probe) was charged with salicylic acid, sodium salt (31.8g, 198 mmol) dissolved in sodium hydroxide (11.9 g, 230 mmol) and water(200 mL). Using an ice bath, the salicylic acid solution was cooled to17° C. and the diazonium salt solution was slowly added in 25 mLportions. Throughout the addition, the pH was maintained at 13.2-13.3with the addition of aqueous sodium hydroxide, and the temperature keptbetween 17-18° C. with the addition of ice. After the addition wascomplete, the resulting dark red solution was allowed to warm to ambienttemperature and stirring was continued for an additional 30 min. Uponacidification to pH 3 with concentrated HCl (˜50 mL, 36.5-38%), a brownsolid precipitated and was collected by suction filtration.

The crude product was purified by flash chromatography (SiO₂:ethylacetate/hexanes, 1:1). On a column packed with 70-230 mesh, 60 Å silicagel with BET surface area of ˜500 m²/g and pore volume of 0.75 cm³/g,the crude product (11.5 g, 38.2 mmol) in DMF (˜12 mL) was loaded.Fractions were collected and combined based on color. The first band wasyellow in color and contained excess salicylic acid as well as traces ofthe desired product. The second band was orange and contained thedesired product, and the third band was red and contained unknownimpurities. All fractions were combined and concentrated under reducedpressure and dried under vacuum.

The purified product was obtained as an orange solid in 28% yield (2.75g): mp 204° C.; ¹H NMR (DMSO-d₆), δ 3.67 (2H,s), 7.11 (1H, d, J=9.0 Hz),7.44 (2H, d, J=8.4 Hz), 7.79 (2H, d, J=8.4 Hz), 8.02 (1H, d of d, J=2.4Hz, 9.0 Hz), 8.29 (1H, s); IR (KBr) 3098, 1696, 1614, 1458, 1345, 1195,838 cm⁻¹; UV-Vis (MeOH), λ_(max)=350 nm, ε=25,700 mol⁻¹ cm⁻¹ L; positiveFAB-MS (NBA), m/z 301 (M+H)⁺, negative FAB-MS(NBA), m/z 299 (M)⁻.

Example 4 Synthesis of 4-(4-Carboxymethyl-Phenylazo)-Phenylacetic Acid

4-Aminophenylacetic acid (3.75 g, 24.8 mmol) was suspended in water (75mL) and concentrated hydrochloric acid (8 mL) was added. The solutionwas cooled to 0° C. in an ice bath with rapid stirring. Sodium nitrite(1.80 g, 26,1 mmol) in water (20 mL) was added dropwise to the4-aminophenylacetic acid solution with rapid stirring. Care was taken tokeep the temperature between 0-5° C. at all times, especially during theNaNO₂ addition. The reaction was stirred for an additional 20 min. Inthe meantime, phenylacetic acid (10.1 g, 74.4 mmol) was dissolved in anaqueous NaOH solution (4.50 g, 113 mmol NaOH in 100 mL H₂O). Thesolution was vigorously stirred at 17° C. and at pH 13.3. The diazoniumsalt solution was added dropwise to the phenylacetic acid solution. Itis of utmost importance to keep the temperature of the phenylacetic acidsolution between 17-18° C. and the pH between 13.2-13.3 at all times,especially during the diazonium salt addition. The temperature wasregulated by the addition of ice and the pH regulated by the addition of8 M NaOH. After addition was complete, the solution was allowed to warmto room temperature and stirred for an additional 30 min. The reactionmixture was suction filtered to remove any undissolved particulates orunwanted side products. The filtrate was acidified with aqueous HCl (10mL conc. HCl in 20 mL H₂O) which produced a dark red precipitate. Theprecipitate was collected by suction filtration and washed several timeswith cold H₂O, until the filtrate was clear. The collected solid was airdried overnight to give the desired compound as a red solid in 37%yield: IR (KBr), 3030 (br), 1696, 1658, 1452, 1414, 1201,850, 675 cm⁻¹FABMS m/z 299 (M+H)⁺, 320 (M+Na); ¹H NMR (DMSO-d₆), δ 3.47 (s, 4H), 7.33(4H, d, J=8.1 Hz), 7.84 (4H, d, J=8.4 Hz).

Example 5 Metabolism of APAZA Following Oral Delivery

The degradation of Apaza(5-(4-carboxymethyl-phenylazo)-2-hydroxy-benzoic acid), a compound ofthe present invention, and sulfasalazine (used as a control; not part ofthe present invention) and the generation of their metabolites whenthese compounds were orally dosed to rats were measured to be able toconfirm that both Apaza and Sulfasalazine undergo bacterial azoreduction and yield their metabolites, 5-aminosalicylic acid (5-ASA) andsulfapyridine for sulfasalazine, 5-aminosalicylic acid (5-ASA) and4-aminophenyl acetic acid (4-APAA) for Apaza.

This experiment was performed to confirm that an azo compound, Apaza,undergoes bacterial reduction process and yields its metabolites inin-vivo metabolism. The quantification of its metabolites was alsocarried out. Sulfasalazine, not part of the present invention, was usedas a control since similar azo bond cleavage by bacteria occurs with it,which results in 5-aminosalicylic acid and sulfapyridine as itsmetabolites. Both Apaza and sulfasalzine were degraded and theirmetabolites were produced as expected.

For urine, the parent compounds and their metabolites were detected withday 1 collection only. The rest of the collections did not show anycompounds. For feces, compounds were detectable up to day 2 collection.

Rats that were dosed with Apaza (rat 1, 2, and 3) showed Apaza, 4-APAA,actarit, and acetylated 5-ASA in urine. Rats with sulfasalazine dosage(rat 4, 5, and 6) showed sulfasalazine, sulfapyridine, and acetylated5-ASA in urine. Only acetylated 5-ASA was detected in feces regardlessof what compounds were given. 5-ASA was quickly converted to acetylated5-ASA.

It is interesting to note that while sulfasalazine dosed rats producedtheir metabolites, 5-ASA (acetylated 5-ASA in this case) andsulfapyridine, in 1:1 ratio, rats with Apaza dosage produced 7 to 10times more of 4-APAA than acetylated 5-ASA.

It is believed that the majority of the ingested sulfasalazine travelsdown the small intestine to the colon and undergoes bacterial azoreduction to liberate sulfapyridine and 5-ASA molecules. The resultsfrom this study confirm this belief and show that Apaza undergoes asimilar bacterial azo reduction.

A total of 8 rats were used for the experiment and methylcellulose wasused as a vehicle. The dosage amount was 100 mg/kg per rat. Three ratswere dosed with Apaza and the other three rats were dosed withsulfasalazine. Two rats were used as a control and dosed withmethylcellulose. Both urine and feces were collected over 4 days andanalyzed by HPLC.

Urine was collected each day and 300 μL of aliquot from each sample wascentrifuged for 10 minutes at 5000 g. 80 μL of supernatant was injectedfor analysis. Feces was also collected each day and homogenized with 1:1mixture of water and acetonitrile. This mixture was then centrifuged for20 minutes at 5000 g. 80 μL of supernatant was injected for analysis.

A Waters 2690 HPLC was used for sample analysis as follows:

Mobile phase programming: Gradient Mobile phase: A = Water + 0.1% TFA B= Acetonitrile + 0.1% TFA Flow rate: 1 mL/min. Column: Phenomenex MaxRP, 80 Å, 4.6 mm × 250 mm PDA settings: Collected spectrum: 210-400 nmExtracted chromatogram: 280 and/or other Run time/sample: Approximately50 min.

Flow Time (mL/minute) % Mobile Phase A % Mobile Phase B — 1 100 0 40 150 50 43 1 5 95 44 1 95 5 50 1 95 5

5-ASA was quickly converted to acetylated 5-ASA. The same amount ofacetylated 5-ASA was generated from both Apaza and sulfasalazine inurine. Acetylated 5-ASA and sulfapyridine were produced in 1:1 ratiofrom sulfasalazine dosed rat urine. Approximately 7 to 10 times more of4-APAA was produced than acetylated 5-ASA from Apaza dosed rat urine.Only acetylated 5-ASA was detected from feces regardless of dosedcompound. More acetylated 5-ASA was detected in feces than urine.

Day 1 Urine Apaza Dosed Total Acetylated Dosage Apaza 4APAA Actarit 5ASA(mg) (mg) (mg) (mg) (mg) Rat 1 22.0 0.48 3.456 0.0717 0.299 Rat 2 23.50.3546 3.177 0.422 Rat 3 22.5 0.4707 4.674 0.298 Urine SulfasalazineDosed Total Acetylated Dosage Sulfasalazine Sulfapyridine 5ASA (mg) (mg)(mg) (mg) Rat 4 21 0.00882 0.337 0.288 Rat 5 22.5 0.01279 0.305 0.328Rat 6 21 0.01092 0.41  0.39  Stool Total Dosage Acetylated (mg) 5ASA(mg) Apaza Dosed Rat 1 22 1.9254 Rat 2 23.5 1.9519 Rat 3 22.5 1.2437Sulfasalazine Dosed Rat 4 21 1.2158 Rat 5 22.5 1.3708 Rat 6 21 0.9033Day 2 Stool Total Dosage Acetylated (mg) 5ASA (mg) Apaza Dosed Rat 1 220.2562 Rat 2 23.5 0.7755 Rat 3 22.5 0.1827 Sulfasalazine Dosed Rat 4 210.2 Rat 5 22.5 0.2584 Rat 6 21 0.1458

Example 6 Biological Effects of Compounds of the Present Invention

The purpose of this study was to histologically evaluate and compare theeffects of three different active pharmaceutical ingredientsadministered intrarectally (twice daily for four days) to male Lewisrats following intrarectal administration of dinitrobenzene sulfonicacid (DNBS). DNBS induced colitis in rats according to an establishedexperimental model (Richard et al., 2000; Bertran et al., 1996; Blau etal., 2000; Kimura et al., 1998; Hogaboam et al., 1996). SHAM and DNBSgroups served as negative and positive controls, respectively. Thedistribution of animals to each group is presented in Table 1:

TABLE 1 GROUP NUMBER OF ANIMALS SHAM 6 DNBS 5 5-ASA 6 4-APAA 6 Mixtureof 5-ASA 4 and 4-APAAMaterials And Methods

Trimmed specimens of colon from 27 male rats were tested, includingmicrotoming, and hematoxylin and eosin staining. The resulting 27 slides(1 transverse section per slide) were examined microscopically. Exceptfor one rat from the SHAM group and one rat from the DNBS group, allslides had their labels taped over to facilitate blind reading. Lesionswere graded on a scale of 1-5 (1=minimal; 2=mild; 3=moderate;4=moderately-severe; 5=severe).

Results

The principal histomorphologic change observed in the colon sections ofall rats treated with DNBS (regardless of any additional treatment) waspartial to full-thickness, fill-length, coagulative-type necrosis.Necrosis was not observed in the saline/methylcellulose treated rats(SHAM group). In all cases, necrotic areas were characterized by adramatic loss of cellular detail and staining affinity; in such areasonly “ghost” outlines of the colonic architecture remained.Occasionally, segmental collapse or “dropout” of an intestinal tissuelayer was evident. Necrotic tissues were heavily invaded by mixed typesof bacteria. In sections that were not completely necrotic, the patternof necrosis tended to be laminar, typically affecting the mucosa andsubmucosa while sparing portions of the muscularis externa and/oraciventitia (serosa and adjacent mesentery). In these sections, a densezone of karyorrhectic neutrophils divided the necrotic inner layers fromthe less affected outer layers. Fibrinoid necrotizing vasculitis ofsubmucosal blood vessels was observed in all DNBS-treated rats.Vasculitis was observed in both necrotic and non-necrotic regions, oftenaccompanied by thrombosis (fibrinous, fibrinocellular, and/or bacterialthrombi), and minimal to moderate submucosal hemorrhage (with or withoutfibrin accumulation). Some hemorrhagic sites contained pigment-ladenmacrophages (siderophages—not separately diagnosed). In all sectionsfrom DNBS-treated rats, the serosa and adjoining mesentery were expandedby mild to moderately severe fibrovascular proliferation (earlygranulation tissue). Sections from two rats (#4 and #11, Mixture of5-ASA and 4-APAA group), each contained a single, short, sharplydemarcated segment of non-necrotic, non-ulcerated mucosa. Changes withinthese comparatively unaffected mucosal segments were limited to minimalto mild crypt epithelial hyperplasia, minimal crypt dilation, andminimal neutrophilic infiltration.

Severity scoring of colonic necrosis was based upon the degree of tissueinvolvement; however, grade 5 (severe) was reserved for lesions in whichnecrosis resulted in extensive tissue loss. Because the pattern ofnecrosis often varied from section to section, the individual intestinallayers were scored separately. Generally, the average severity scoresfor necrosis were comparable among the four groups of DNBS-treated rats(Table 2). The average score for mucosal necrosis in the Mixture of5-ASA and 4-APAA group was lower than scores in the other groups ofDNBS-treated rats due to the spared areas of mucosa in two animals fromthe Mixture of 5-ASA and 4-APAA group.

TABLE 2 Average Tissue Necrosis Scores Mixture 5-ASA & Group SHAM DNBS5-ASA 4-APAA 4-APAA No. Animals (6) (5) (6) (6) (4) Mucosa 0.00 4.204.50 4.33 3.50 Submucosa 0.00 4.20 4.17 4.00 4.25 Muscularis 0.00 3.603.5  3.17 3.00 Adventitia 0.00 1.40 1.67 1.67 1.50

SUMMARY

The principal histomorphologic change observed in the colon sections ofall rats treated with DNBS (regardless of any additional treatment) waspartial to full-thickness, full-length, coagulative-type necrosis.Associated changes included massive bacterial invasion of the necrotictissue, fibrinoid necrotizing vasculitis with thrombosis and hemorrhage,and heavy neutrophilic infiltration. Necrosis was not observed in thesaline/methylcellulose-treated rats (SHAM group). The severity (extent)of necrosis was comparable among the four groups of DNBS-treated rats(DNBS, 5-ASA, 4-APAA, and Mixture of 5-ASA and 4-APAA), except thatsingle segments of mucosa were comparatively spared in 2/4 rats from theMixture of 5-ASA and 4-APAA group.

Example 7 Anti-Inflammatory Activity of Drug Mixture

Dinitrobenzene sulfonic acid (DNBS) colitis was induced (no etheranesthesia) in 4 groups of 6 Lewis rats each. One DNBS group was dosedwith vehicle (0.7% methyl cellulose) as well as an additional sham groupof 6 animals that received a saline enema instead of DNBS. Intrarectal(ir) dosing was performed in conscious animals b.i.d-for 4 days. Drugtreatments were as follows:

5-aminosalicylic acid (5-ASA): 50 mg/kg

4aminophenylacetic acid (4-APAA): 49.5 mg/kg (equimolar to 5-ASA)

Mixture: 5-ASA+4-APAA: 50 mg/kg+49.5 mg/kg

Drugs were suspended in the above mentioned vehicle and staff blinded todrug groups. Daily weights and diarrhea scores were recorded. On the 5thday post-irritant rats were sacrificed, laparotomies performed andscored for intestinal adhesions and strictures; colectomized and colonweights recorded, colons opened longitudinally andinflammation/ulcerations scored.

Results illustrated in FIGS. 3 and 4 indicated that 5-ASA, 4-APAA, andthe mixture produce similar anti-inflammatory activity (˜31% reductionin colon:body weight [% BW]). The severity of inflammation approachedmaximum. It is possible that the severity could be titrated by reductionof the DNBS dose and a small study was performed to test thishypothesis. It is possible that with a milder insult there may beevidence of greater separation of treatment effects.

DNBS colitis was induced in 6 Lewis rats (3 at 30 and 3 at 15 mg/ratDNBS) and allowed to develop for 5 days with no treatment in order tocitrate the severity of inflammation. Diarrhea was noted on days 1-4 andthe rats were sacrificed on day 5, scored, and colon:body weightdetermined. Results indicate that 15 mg/rat DNBS produces milder butinconsistent inflammation compared to 30 mg. The 30 mg/kg DNBS resultwas consistent with that seen previously.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A method of treating an inflammatory condition of the GI tract in asubject comprising administering to a subject in need of such treatmentan effective amount of an active pharmaceutical ingredient comprising acompound of Formula II:

where R⁹, R¹⁰ and R¹¹ are independently selected from the groupconsisting of hydrogen and C₁ to C₄ alkyl; and R¹² is selected from thegroup consisting of hydrogen and —C(O)R¹³, where R¹³ is a C₁ to C₆ alkylor an aryl group; or an ester or a pharmaceutically acceptable salt ofsuch compound, in admixture with pharmaceutical diluent or carrier. 2.The method according to claim 1, wherein the active pharmaceuticalingredient further comprises a compound of Formula (III):

or an ester or a pharmaceutically acceptable salt thereof.
 3. The methodaccording to claim 1, wherein the compound of Formula II is from about10 to 90 weight percent of the active pharmaceutical ingredient.
 4. Themethod according to claim 1, wherein the molar ratio of the compound ofFormula II to the compound of Formula III is between about 1:10 and10:1.
 5. The method according to claim 1, wherein R⁹, R¹⁰, and R¹¹ areindependently selected from the group consisting of H, CH₃, CH₂CH3, andCH(CH₃)₂.
 6. The method according to claim 1, wherein R⁹, R¹⁰, and R¹¹are independently selected from the group consisting of H, and CH₃. 7.The method according to claim 1, wherein R¹² is hydrogen.
 8. An in vivomethod of generating active metabolites for the treatment of aninflammatory condition of the GI tract, the method comprising orallyadministering to a subject having intestinal bacterial a compound havingthe structure:

5-(4-carboxymethylphenylazo)-2-hydroxy-benzoic acid or an ester or apharmaceutically acceptable salt thereof, wherein the active metabolitescomprise at least one compound selected from the group consisting of5-aminosalicylic acid (5-ASA) and 4-aminophenyl acetic acid (4-APAA). 9.The in vivo method of claim 8, wherein the active metabolite furtherincludes acetylated 5-ASA and acetylated 4-APAA.
 10. The in vivo methodof claim 8, wherein the 5-(4-carboxymethylphenylazo)-2-hydroxy-benzoicacid or an ester or a pharmaceutically acceptable salt thereof is in aliquid solution comprising a biocompatible carrier.
 11. The in vivomethod of claim 8, wherein the5-(4-carboxymethylphenylazo)-2-hydroxy-benzoic acid or an ester or apharmaceutically acceptable salt thereof is in a powder form.
 12. The invivo method of claim 8, wherein the inflammatory condition of the GItract is ulcerative colitis.
 13. The in vivo method of claim 8, whereinthe inflammatory condition of the GI tract is Crohn's disease.
 14. Thein vivo method of claim 8, wherein the active metabolites were generatedwithin 24 hours of administration.
 15. The in vivo method of claim 14,wherein the active metabolites were located in urine or feces matter ofsubject.
 16. A liquid solution comprising:5-(4-carboxymethylphenylazo)-2-hydroxy-benzoic acid, 5-aminosalicylicacid (5-ASA) and 4-aminophenyl acetic acid (4-APAA).
 17. The liquidsolution of claim 16 further comprising 4-aminophenyl acetic acid(4-APAA), acetylated 5-ASA and acetylated 4-APAA.
 18. A method forgenerating active metabolites; the method comprising contactingintestinal bacteria with 5-(4-carboxymethylphenylazo)-2-hydroxy-benzoicacid or an pharmaceutically acceptable salt thereof under conditions formetabolism thereof by the bacteria.
 19. The method of claim 18, whereinthe active metabolites comprises at least one metabolite selected fromthe group consisting of 5-aminosalicylic acid (5-ASA) and 4-aminophenylacetic acid (4-APAA).
 20. The method of claim 19, further comprising4-aminophenyl acetic acid (4-APAA), acetylated 5-ASA and acetylated4-APAA.